Germ cell-specific expression of green fluorescent protein in transgenic rainbow trout under control of the rainbow trout vasa-like gene promoter

Abstract

A technique to identify and isolate live fish primordial germ cells (PGCs) has not been established, in spite of the importance of purified germ cells for molecular and cellular studies. In rainbow trout, the distribution of vasa transcripts is restricted to the germ cell lineage, making this transcript a useful indicator of PGCs. Therefore, in this study, we cloned and characterized the rainbow trout vasa-like gene (RtVLG) regulatory regions and produced transgenic trout carrying the green fluorescent protein (GFP) gene driven by the RtVLG regulatory regions (p vasa-GFP) in order to identify live PGCs in vivo. In transgenic trout carrying the p vasa-GFP construct, cells showing green fluorescence were first observed at the mid-blastula stage; however, no cell-type-specific expression was observed at this stage. At the eyed stage, about 30% of the transgenic embryos showed specific GFP expression in PGCs, and at the hatching stage, about 70% of the transgenic embryos did so. An immunohistochemical study of hatching stage embryos revealed that the GFP-expressing cells are located in genital ridges. This transgenic trout, having visualizable PGCs, will make it possible to isolate live PGCs for in vitro studies and to study the ontogeny of PGCs including sex differentiation in live embryos.